Membrane-bound carbonic anhydrase (CA) facilitates acidification in the kidney. While most hydratase activity is considered due to CA IV, some in the basolateral membranes could be attributed to CA XII. Indeed, CA IV is glycosylphosphatidylinositol-anchored, connoting apical polarization, but CA IV immunoreactivity has been detected on basolateral membranes of proximal tubules. Herein, we determined whether CA XII mRNA was expressed in acidifying segments of the rabbit nephron. The open reading frame of CA XII was sequenced from a rabbit kidney cortex cDNA library; it was 83% identical to human CA XII and coded for a 355 amino acid single-pass transmembrane protein. Northern blot revealed an abundant 4.5 kb message in kidney cortex, medulla and colon. By in situ hybridization CA XII mRNA was expressed by proximal convoluted and straight tubules, cortical and medullary collecting ducts, and papillary epithelium. By RT-PCR CA XII mRNA was abundantly expressed in cortical and medullary collecting ducts and thick ascending limb of Henle's loop; it was also expressed in proximal convoluted and straight tubules but not in glomeruli or S3 segments. FLAG-CA XII of ~40 kDa expressed in E. coli showed hydratase activity that was inhibited by 0.1 mM acetazolamide. Unlike CA IV, expressed CA XII activity was inhibited by 1% SDS, suggesting insufficient disulfide linkages to stabilize the molecule. Western blotting of expressed CA XII using two anti-rabbit CA IV peptide antibodies showed no cross-reactivity. Our findings indicate that CA XII may contribute to the membrane CA activity of proximal tubules and collecting ducts.